Urinary extracellular vesicle–associated IL-6 and cathepsin D for early identification of chronic kidney disease in high-risk patients

ABSTRACT Background: Early chronic kidney disease (CKD) is frequently asymptomatic and remains underdiagnosed in high-risk populations. Urinary extracellular vesicles (uEVs) provide a non-invasive source of kidney-related biomolecules and may improve early detection strategies. We evaluated uEV-associated interleukin-6 (IL-6), cathepsin D (CTD), and transforming growth factor-β (TGF-β) as candidate biomarkers for early CKD, including sex-stratified patterns. Methods: In this cross-sectional study, 230 adults at high risk (type 2 diabetes mellitus and/or systemic arterial hypertension) were classified as having normal kidney function (NKF) or CKD stages G1–G4. uEVs were isolated using a standardized protocol and characterized by scanning electron microscopy, Raman spectroscopy, dynamic light scattering, and Western blot for Alix and flotillin-1. Total uEV-associated protein was quantified, and CTD, IL-6, and TGF-β were measured in uEV preparations. Results: uEVs displayed expected morphology and marker expression. Particle size distributions differed by sex and CKD status. Total uEV protein content did not significantly differ across CKD stages. uEV-associated IL-6 was significantly higher in G1 compared with NKF (p ≤ 0.05). CTD showed a decreasing trend from G1 to G4 with stage- and sexspecific differences, most evident in women at G1. uEV-associated TGF-β did not differ significantly across stages. Conclusions: uEV-derived IL-6 and CTD show potential as non-invasive indicators of early CKD, with clinically relevant sex-dependent patterns, while TGF-β may provide complementary information. Standardized uEV isolation and measurement workflows are essential for biomarker reproducibility and future clinical translation.